The goal of this proposal is to understand the related processes of secretory protein transport and lipid metabolism in African trypanosomes. These processes have been defined primarily in vertebrate tissue culture cells and yeast. However, the availability of sophisticated genetic strategies for manipulation of Trypanosoma brucei provides a potent system in which to study general cell biology. More importantly, two features make African trypanosomes particularly attractive for study. First, trypanosomes are high impact human and veterinary pathogens in sub-Saharan Africa. The WHO currently estimates that 60 million people in 36 countries are at risk of acquiring Human African Trypanosomiasis. Only a handful of drugs are in use for treatment, the best of which (eflornithine) is expensive and requires a difficult regimen, the worst of which (melarsoprol) kills up to 10% of recipients. Infection is inevitably fatal without intervention, and since vaccination is not an option there is a critical need for new drug development. Second, the phylogenetically ancient relationship of trypanosomes to other eukaryotes ensures that whatever results are obtained are as likely to be of interest for their commonality with, as for their distinctness from, standard 'higher' eukaryotic systems. The lynchpin of pathogenesis in these parasites is the glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) of the bloodstream stage of the life cycle. Understanding how VSG, and related transferrin receptor, are transported to and maintained at the cell surface, and the role of GPI anchors in proper trafficking, are critical to understanding the parasite half of the host- parasite relationship. All of the Specific Aims in this application for competitive renewal derive directly from progress made during the current funding period. First, we have confirmed that GPI valence regulates progression and ultimate stability of proteins within the secretory pathway. In Aim #1 we will continue these studies by defining the default route of GPI-minus reporters to the lysosome, and by investigating the apparently aberrant behavior of native transferrin receptor. Second, we have demonstrated that VSG is selectively loaded into COPII vesicles for ER exit. In Aim #2 we will study the machinery of GPI-dependent targeting in the early secretory pathway of trypanosomes. Third, in our studies of the role of parasite lipids in GPI-dependent trafficking we have found that sphingomyelin synthesis is essential in bloodstream stage trypanosomes, in contrast to other kinetoplasted protozoa, which do not make sphingomyelin at all. In Aim #3 we will complete our characterization of a unique family of sphingolipid synthases using a novel and highly innovative cell-free system for synthesis of membrane proteins. These studies will broaden our knowledge of basic cell biological processes in all eukaryotes, and will lay the groundwork for targeting the sphingolipid pathway for novel drug development.